scholarly journals Gating Dynamics of the Acetylcholine Receptor Extracellular Domain

2004 ◽  
Vol 123 (4) ◽  
pp. 341-356 ◽  
Author(s):  
Sudha Chakrapani ◽  
Timothy D. Bailey ◽  
Anthony Auerbach
Keyword(s):  
Conformational Change ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Extracellular Domain ◽  
Mouse Muscle ◽  
Α Subunit ◽  
Acetylcholine Binding Protein ◽  
Almost All ◽  
Loop 2 ◽  
Sandwich Core

We used single-channel recording and model-based kinetic analyses to quantify the effects of mutations in the extracellular domain (ECD) of the α-subunit of mouse muscle–type acetylcholine receptors (AChRs). The crystal structure of an acetylcholine binding protein (AChBP) suggests that the ECD is comprised of a β-sandwich core that is surrounded by loops. Here we focus on loops 2 and 7, which lie at the interface of the AChR extracellular and transmembrane domains. Side chain substitutions in these loops primarily affect channel gating by either decreasing or increasing the gating equilibrium constant. Many of the mutations to the β-core prevent the expression of functional AChRs, but of the mutants that did express almost all had wild-type behavior. Rate-equilibrium free energy relationship analyses reveal the presence of two contiguous, distinct synchronously-gating domains in the α-subunit ECD that move sequentially during the AChR gating reaction. The transmitter-binding site/loop 5 domain moves first (Φ = 0.93) and is followed by the loop 2/loop 7 domain (Φ = 0.80). These movements precede that of the extracellular linker (Φ = 0.69). We hypothesize that AChR gating occurs as the stepwise movements of such domains that link the low-to-high affinity conformational change in the TBS with the low-to-high conductance conformational change in the pore.

scholarly journals The Extracellular Linker of Muscle Acetylcholine Receptor Channels Is a Gating Control Element

2000 ◽  
Vol 116 (3) ◽  
pp. 327-340 ◽  
Author(s):  
Claudio Grosman ◽  
Frank N. Salamone ◽  
Steven M. Sine ◽  
Anthony Auerbach
Keyword(s):  
Acetylcholine Receptors ◽  
Single Channel ◽  
Channel Gating ◽  
Congenital Myasthenic Syndrome ◽  
Control Element ◽  
Dependent Manner ◽  
Α Subunit ◽  
Transmembrane Segments ◽  
Channel Opening ◽  
Myasthenic Syndrome

We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2–M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the α subunit (αS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the αS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an ∼92-fold increased gating equilibrium constant, which is consistent with an ∼10-fold decreased EC50 in the presence of ACh. With choline, this mutation accelerates channel opening ∼28-fold, slows channel closing ∼3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, αS269I acetylcholine receptors open at a rate of ∼1.4 × 106 s−1 and close at a rate of ∼760 s−1. These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of ∼140 s−1. Ile mutations at positions flanking αS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the α subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the αS269I mutation, Ile mutations at equivalent positions of the β, ε, and δ subunits do not affect apparent open-channel lifetimes. However, in β and ε, shifting the mutation one residue to the NH2-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2–M3L of the α subunit moves before the corresponding linkers of the β and ε subunits.

scholarly journals Efficiency measures the conversion of agonist binding energy into receptor conformational change

2019 ◽  
Vol 151 (4) ◽  
pp. 465-477 ◽  
Author(s):  
Tapan K. Nayak ◽  
Ridhima Vij ◽  
Iva Bruhova ◽  
Jayasha Shandilya ◽  
Anthony Auerbach
Keyword(s):  
Activation Energy ◽  
Binding Energy ◽  
Conformational Change ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Equilibrium Constants ◽  
Bk Channels ◽  
Agonist Binding ◽  
Aryl Hydrocarbon ◽  
Efficiency Measures

Receptors alternate between resting↔active conformations that bind agonists with low↔high affinity. Here, we define a new agonist attribute, energy efficiency (η), as the fraction of ligand-binding energy converted into the mechanical work of the activation conformational change. η depends only on the resting/active agonist-binding energy ratio. In a plot of activation energy versus binding energy (an “efficiency” plot), the slope gives η and the y intercept gives the receptor’s intrinsic activation energy (without agonists; ΔG0). We used single-channel electrophysiology to estimate η for eight different agonists and ΔG0 in human endplate acetylcholine receptors (AChRs). From published equilibrium constants, we also estimated η for agonists of KCa1.1 (BK channels) and muscarinic, γ-aminobutyric acid, glutamate, glycine, and aryl-hydrocarbon receptors, and ΔG0 for all of these except KCa1.1. Regarding AChRs, η is 48–56% for agonists related structurally to acetylcholine but is only ∼39% for agonists related to epibatidine; ΔG0 is 8.4 kcal/mol in adult and 9.6 kcal/mol in fetal receptors. Efficiency plots for all of the above receptors are approximately linear, with η values between 12% and 57% and ΔG0 values between 2 and 12 kcal/mol. Efficiency appears to be a general attribute of agonist action at receptor binding sites that is useful for understanding binding mechanisms, categorizing agonists, and estimating concentration–response relationships.

scholarly journals The Role of Loop 5 in Acetylcholine Receptor Channel Gating

2003 ◽  
Vol 122 (5) ◽  
pp. 521-539 ◽  
Author(s):  
Sudha Chakrapani ◽  
Timothy D. Bailey ◽  
Anthony Auerbach
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Single Channel ◽  
Channel Gating ◽  
Α Subunit ◽  
Acetylcholine Binding Protein ◽  
Relationship Analysis ◽  
Subunit Interface ◽  
Acetylcholine Receptor Channel ◽  
Receptor Channel

Nicotinic acetylcholine receptor channel (AChR) gating is an organized sequence of molecular motions that couples a change in the affinity for ligands at the two transmitter binding sites with a change in the ionic conductance of the pore. Loop 5 (L5) is a nine-residue segment (mouse α-subunit 92–100) that links the β4 and β5 strands of the extracellular domain and that (in the α-subunit) contains binding segment A. Based on the structure of the acetylcholine binding protein, we speculate that in AChRs L5 projects from the transmitter binding site toward the membrane along a subunit interface. We used single-channel kinetics to quantify the effects of mutations to αD97 and other L5 residues with respect to agonist binding (to both open and closed AChRs), channel gating (for both unliganded and fully-liganded AChRs), and desensitization. Most αD97 mutations increase gating (up to 168-fold) but have little or no effect on ligand binding or desensitization. Rate-equilibrium free energy relationship analysis indicates that αD97 moves early in the gating reaction, in synchrony with the movement of the transmitter binding site (Φ = 0.93, which implies an open-like character at the transition state). αD97 mutations in the two α-subunits have unequal energetic consequences for gating, but their contributions are independent. We conclude that the key, underlying functional consequence of αD97 perturbations is to increase the unliganded gating equilibrium constant. L5 emerges as an important and early link in the AChR gating reaction which, in the absence of agonist, serves to increase the relative stability of the closed conformation of the protein.

scholarly journals Modal affinities of endplate acetylcholine receptors caused by loop C mutations

2015 ◽  
Vol 146 (5) ◽  
pp. 375-386 ◽  
Author(s):  
Ridhima Vij ◽  
Prasad Purohit ◽  
Anthony Auerbach
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptors ◽  
Time Course ◽  
Single Channel ◽  
Equilibrium Constants ◽  
Open Probability ◽  
In The Wild ◽  
Single Channel Currents ◽  
Almost All ◽  
Channel Currents

The time course of the endplate current is determined by the rate and equilibrium constants for acetylcholine receptor (AChR) activation. We measured these constants in single-channel currents from AChRs with mutations at the neurotransmitter-binding sites, in loop C. The main findings are: (a) Almost all perturbations of loop C generate heterogeneity in the channel open probability (“modes”). (b) Modes are generated by different affinities for ACh that can be either higher or lower than in the wild-type receptors. (c) The modes are stable, in so far as each receptor maintains its affinity for at least several minutes. (d) Different agonists show different degrees of modal activity. With the loop C mutation αP197A, there are four modes with ACh but only two with partial agonists. (e) The affinity variations arise exclusively from the αδ-binding site. (f) Substituting four γ-subunit residues into the δ subunit (three in loop E and one in the β5–β5′ linker) reduces modal activity. (g) At each neurotransmitter-binding site, affinity is determined by a core of five aromatic residues. Modes are eliminated by an alanine mutation at δW57 but not at the other aromatics. (h) Modes are eliminated by a phenylalanine substitution at all core aromatics except αY93. The results suggest that, at the αδ agonist site, loop C and the complementary subunit surface can each adopt alternative conformations and interact with each other to influence the position of δW57 with respect to the aromatic core and, hence, affinity.

scholarly journals Acetylcholine Receptor Gating: Movement in the α-Subunit Extracellular Domain

2007 ◽  
Vol 130 (6) ◽  
pp. 569-579 ◽  
Author(s):  
Prasad Purohit ◽  
Anthony Auerbach
Keyword(s):  
Acetylcholine Receptor ◽  
Single Channel ◽  
Equilibrium Constants ◽  
Extracellular Domain ◽  
Α Subunit ◽  
Subunit Interface ◽  
Channel Opening ◽  
Affinity Change ◽  
A Subunit ◽  
Single Channel Currents

Acetylcholine receptor channel gating is a brownian conformational cascade in which nanometer-sized domains (“Φ blocks”) move in staggering sequence to link an affinity change at the transmitter binding sites with a conductance change in the pore. In the α-subunit, the first Φ-block to move during channel opening is comprised of residues near the transmitter binding site and the second is comprised of residues near the base of the extracellular domain. We used the rate constants estimated from single-channel currents to infer the gating dynamics of Y127 and K145, in the inner and outer sheet of the β-core of the α-subunit. Y127 is at the boundary between the first and second Φ blocks, at a subunit interface. αY127 mutations cause large changes in the gating equilibrium constant and with a characteristic Φ-value (Φ = 0.77) that places this residue in the second Φ-block. We also examined the effect on gating of mutations in neighboring residues δI43 (Φ = 0.86), εN39 (complex kinetics), αI49 (no effect) and in residues that are homologous to αY127 on the ε, β, and δ subunits (no effect). The extent to which αY127 gating motions are coupled to its neighbors was estimated by measuring the kinetic and equilibrium constants of constructs having mutations in αY127 (in both α subunits) plus residues αD97 or δI43. The magnitude of the coupling between αD97 and αY127 depended on the αY127 side chain and was small for both H (0.53 kcal/mol) and C (−0.37 kcal/mol) substitutions. The coupling across the single α–δ subunit boundary was larger (0.84 kcal/mol). The Φ-value for K145 (0.96) indicates that its gating motion is correlated temporally with the motions of residues in the first Φ-block and is not synchronous with those of αY127. This suggests that the inner and outer sheets of the α-subunit β-core do not rotate as a rigid body.

scholarly journals Acetylcholine Receptor Gating at Extracellular Transmembrane Domain Interface: the “Pre-M1” Linker

2007 ◽  
Vol 130 (6) ◽  
pp. 559-568 ◽  
Author(s):  
Prasad Purohit ◽  
Anthony Auerbach
Keyword(s):  
Acetylcholine Receptors ◽  
State Energy ◽  
Transmembrane Domain ◽  
Salt Bridge ◽  
Side Chain ◽  
Coupling Energy ◽  
And Function ◽  
Loop 2 ◽  
Α Subunits ◽  
Α1 Subunit

Charged residues in the β10–M1 linker region (“pre-M1”) are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational “wave.” We examined the effects of mutations to all five residues in pre-M1 (positions M207–P211) plus E45 in loop 2 in the mouse α1-subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant (Keq), but the changes for R209, L210, and E45 were larger. Of 19 different side chain substitutions at R209 on the wild-type background, only Q, K, and H generated functional channels, with the largest change in Keq (67-fold) from R209Q. Various R209 mutants were functional on different E45 backgrounds: H, Q, and K (E45A), H, A, N, and Q (E45R), and K, A, and N (E45L). Φ values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Φ values for R209 on the wt and three other backgrounds could not be estimated because of scatter. The average coupling energy between 209/45 side chains (six different pairs) was only −0.33 kcal/mol (for both α subunits, combined). Pre-M1 residues are important for expression of functional channels and participate in gating, but the relatively modest changes in closed- vs. open-state energy caused mutations, the weak coupling energy between these residues and the functional activity of several unmatched-charge pairs are not consistent with the perturbation of a salt bridge between R209 and E45 playing the principle role in gating.

scholarly journals The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions

2010 ◽  
Vol 136 (1) ◽  
pp. 63-82 ◽  
Author(s):  
Natascia Vedovato ◽  
David C. Gadsby
Keyword(s):  
Point Mutations ◽  
Pump Current ◽  
Binding Pocket ◽  
Inhibitory Influence ◽  
Electrical Measurements ◽  
Α Subunit ◽  
Apparent Affinity ◽  
C Terminus ◽  
Na Ions ◽  
Almost All

Interactions of the three transported Na ions with the Na/K pump remain incompletely understood. Na/K pump crystal structures show that the extended C terminus of the Na,K–adenosine triphosphatase (ATPase) α subunit directly contacts transmembrane helices. Deletion of the last five residues (KETYY in almost all Na/K pumps) markedly lowered the apparent affinity for Na activation of pump phosphorylation from ATP, a reflection of cytoplasmic Na affinity for forming the occluded E1P(Na3) conformation. ATPase assays further suggested that C-terminal truncations also interfere with low affinity Na interactions, which are attributable to extracellular effects. Because extracellular Na ions traverse part of the membrane’s electric field to reach their binding sites in the Na/K pump, their movements generate currents that can be monitored with high resolution. We report here electrical measurements to examine how Na/K pump interactions with extracellular Na ions are influenced by C-terminal truncations. We deleted the last two (YY) or five (KESYY) residues in Xenopus laevis α1 Na/K pumps made ouabain resistant by either of two kinds of point mutations and measured their currents as 10-mM ouabain–sensitive currents in Xenopus oocytes after silencing endogenous Xenopus Na/K pumps with 1 µM ouabain. We found the low affinity inhibitory influence of extracellular Na on outward Na/K pump current at negative voltages to be impaired in all of the C-terminally truncated pumps. Correspondingly, voltage jump–induced transient charge movements that reflect pump interactions with extracellular Na ions were strongly shifted to more negative potentials; this signals a several-fold reduction of the apparent affinity for extracellular Na in the truncated pumps. Parallel lowering of Na affinity on both sides of the membrane argues that the C-terminal contacts provide important stabilization of the occluded E1P(Na3) conformation, regardless of the route of Na ion entry into the binding pocket. Gating measurements of palytoxin-opened Na/K pump channels additionally imply that the C-terminal contacts also help stabilize pump conformations with occluded K ions.

Some properties of acetylcholine receptors in human cultured myotubes

1985 ◽  
Vol 224 (1235) ◽  
pp. 183-196 ◽  
Keyword(s):  
Acetylcholine Receptors ◽  
Single Channel ◽  
Fold Increase ◽  
Resting Potential ◽  
Patch Clamp Technique ◽  
Membrane Hyperpolarization ◽  
Human Myotubes ◽  
Cultured Myotubes ◽  
Channel Open Time ◽  
Ach Receptors

The distribution and single channel properties of acetylcholine (ACh) receptors in human myotubes grown in tissue culture have been examined. Radioautography of myotubes labelled with [ 125 I]α-bungarotoxin showed that ACh receptors are distributed uniformly over the myotube surface at a density of 3.9 ± 0.5 receptors per square micrometre. Ac­cumulations of ACh receptors (hot spots) were found rarely. The conductance and kinetics of ACh-activated channels were investi­gated with the patch-clamp technique. Cell-attached membrane patches were used in all experiments. A single channel conductance in the range 40–45 pS was calculated. No sublevels of conductance (substates) of the activated channel were observed. The distribution of channel open-times varied with ACh concentration. With 100 nM ACh, the distribution was best fitted by the sum of two exponentials, whereas with 1 μM ACh a single exponential could be fitted. The mean channel open-time at the myotube resting potential (ca. — 70 mV, 22°C) was 8.2 ms. The distribution of channel closed-times was complex at all concentrations of ACh studied (100 nM to 10 μm). With desensitizing doses of ACh (10 μM), channel openings occurred in obvious bursts; each burst usually appeared as part of a ‘cluster’ of bursts. Both burst duration and mean interval between bursts increased with membrane hyperpolarization. Individual channel open-times and burst durations showed similar voltage dependence (e-fold increase per 80 mV hyperpolarization), whereas both the channel closed-times within a burst and the number of openings per burst were independent of membrane potential.

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